Congenital heart disease is the most common type of birth defect and is the leading non-infectious cause of death in the first year of life. Malformations of the aortic valves are arguably the most common type of cardiac malformation as bicuspid aortic valve alone has an estimated prevalence of 1-2% in the population. The mechanisms underlying the development of aortic valve abnormalities are not well understood. We were the first to report that heterozygous mutations in NOTCH1 were associated with bicuspid aortic valve in humans but there remains a major void in our understanding of the mechanisms by which aortic valve malformations occur. We have generated a new mouse model of highly penetrant aortic valve malformations (bicuspid aortic valve) using Notch1 heterozygote mice backcrossed into a Nos3 (endothelial nitric oxide synthase)-null background. These mice display a near 100% incidence of aortic valve abnormalities including thickened, bicuspid and dysfunctional aortic valves. Our preliminary studies suggest that Nos3 genetically interacts with Notch1 in the valve endothelium to cause valve stenosis. Additional studies suggest that loss of Nos3 inhibits Notch1 signaling in endothelial cells to cause valve defects by an epigenetic mechanism. The overall hypothesis is that deficiency of Notch1 in endothelial cell lineages leads to BAV by disrupting the remodeling of developing aortic valve cushion mesenchyme. In this proposal, we will elucidate the cellular and molecular abnormalities in this clinically relevant model of aortic valve malformations. The specific aims of the proposal are: Specific Aim 1. To define the cellular and molecular mechanisms underlying the development of bicuspid aortic valve. Specific Aim 2. To determine the cell lineage requirement for Notch1 signaling for normal aortic valve morphogenesis. Specific Aim 3. To determine the mechanisms by which nitric oxide regulates Notch1 in the remodeling aortic valve.